Carbohydrate Attachment Research Articles

Cloning and nucleotide sequence of mouse immunoglobulin epsilon chain cDNA.

cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.

Structural studies on equine chorionic gonadotropin

The amino acid sequence of the α subunit of equine chorionic gonadotropin (eCG, also pregnant mare serum gonadotropin, PMSG) has been determined. Overlapping peptides from tryptic and chymotrypic digests were isolated by a two-dimensional peptide mapping technique and sequenced by the Edman procedure. The proposed amino acid sequence of eCG α is: (**Denotes carbohydrate attachment points.) This sequence differs significantly from that proposed by Rathnamet al. (1978) for equine follitropin α subunit; in particular, their sequence lacked the first fourteen residues.

Complete amino acid sequence of a mouse mu chain: homology among heavy chain constant region domains.

The complete amino acid sequence of the mouse mu chain secreted by the MOPC 104E myeloma tumor has been determined. There are four constant region domains in the mu chain and a 20-residue COOH-terminal segment that plays a role in the polymerization of pentameric immunoglobulin M molecules. There are six sites of carbohydrate attachment in the MOPC 104E mu chain. Three complex-type and two high-mannose oligosaccharides are located in the mu chain constant region. The general type and location of carbohydrate moieties in the mu chain constant region are completely conserved between mouse and human mu chains. Homology in the location of carbohydrate structures on different classes of heavy chains is discussed.

The effects of tunicamycin on the synthesis and export of fat body proteins and glycoproteins in larvae of the greater wax moth Galleria mellonella (L.)

Treatment of fat body from the greater wax moth, Galleria mellonella (L.), with tunicamycin in vitro caused complex alterations in protein synthesis. These included a 50% inhibition of protein synthesis and a greater than 90% inhibition of both protein release and glycoproteins biosyntheis. The inhibition of glycoprotein biosynthesis can be accounted for by the well-documented effects of tunicamycin on GlcNAc-pyrophosphoryl dolichol synthesis, while the two additional effects are likely to have a complex basis. Synthesis and export of the four storage proteins was also studied in fat body treated with tunicamycin. Exposure to antibiotic for short periods of time (2.5 hr) resulted in the sysnthesis of lower mol. wt variants of at least two of these polypeptides in treated cultures, both of which normally contain carbohydrate. Treated fat body released only putative fully-glycosylated forms of the storage proteins which suggests that the attachment of carbohydrate is in one way necessary for their secretion. Newly synthesized storage proteins were not detected in fat body treated with tunicamycin for longer time periods (approx. 7 hr), which indicates carbohydrate may also increase the intracellular half life of this class of proteins.

Amino acid sequence of carboxypeptidase Y. II. Peptides from enzymatic cleavages

Carboxypeptidase Y (E.C.3.4.12.1) with the lysine side-chains blocked by citraconylation has been digested with trypsin and the resulting peptides purified by gel filtration followed by ion-exchange chromatography on DE 52 cellulose. Eight of the expected ten peptides have been completely or partially sequenced in a liquid phase automatic sequencer. The remaining two peptides were sequenced as part of a CNBr peptide (see preceeding paper). Selected peptides obtained from enzymatic cleavages with A. mellea protease, chymotrypsin, post-proline cleaving enzyme, S. aureus V8 protease, and subtilisin have upon sequencing provided overlaps which have made the reconstruction of 96% of the complete sequence possible together with a tentative assignment of the carbohydrate attachment sites. No homology with sequences of known proteases has been observed.

37] Preparation of peptides from bovine rhodopsin

This chapter discusses the preparation of peptides from bovine rhodopsin. Rhodopsin's carboxyl-terminal cyanogen bromide peptide (CB3) contains the protein's phosphorylation sites. It is preceded in the sequence by a cysteine-containing cyanogen bromide peptide, CB-6. This cysteine reacts with N -ethylmaleimide and iodoacetamidosalicylate in the dark and is probably the site for reaction of fluorescent and spin-label alkylating reagents. Membrane-bound rhodopsin may be subjected to limited proteolysis by thermolysin, yielding two large membrane-bound peptide fragments, F1 and F2. Chromatographically pure lipid-free rhodopsin is harvested by centrifugation following dialysis to remove detergent. The amino-terminal tryptic glycopeptide of rhodopsin (T1), which contains the protein's two sites of carbohydrate attachment, has been prepared and its amino acid sequence and carbohydrate structure determined.

A monoclonal immunoglobulin G1 in which some molecules possess glycosylated light chains—I. Site of glycosylation

A human monoclonal IgG1 κ protein (Hom) was found to possess two species of light chain with molecular weights of 23,000 and 28,000 respectively. The difference in mass was due to the presence of a carbohydrate prosthetic group on the 28,000 dalton species. The two species of light chain were separated by chromatography on CM-cellulose. Circular dichroism failed to detect any significant differences in conformation between the glycosylated and non-glycosylated forms and both species recombined with a heavy chain in an identical fashion as judged by difference spectroscopy. An anti-idiotypic antibody raised against the glycosylated Fab-fragment of IgGlHom was unable to distinguish between this species and the non-glycosylated Fab fragment, suggesting that the structure of the combining site is identical or very similar in both molecules. Amino-acid sequence analyses showed that both light chains had an identical sequence up to residue 41. Further studies on the glycosylated light chain, using o-iodosobenzoic acid fragments, localized the carbohydrate attachment site to asparagine 107, the penultimate residue of the J κ region which links V κ to C κ. The sequence around the attachment site was Asn-Arg-Thr which satisfies the requirements for an acceptor sequence. The non-glycosylated light chain was found to have the same sequence in this region indicating that the absence of glycosylation was not due to the lack of an acceptor sequence. A kinetic mechanism has been proposed to account for the incomplete glycosylation of the light chains of IgG1Hom in which there is a competition between the rate of folding of the nascent polypeptide and the rate of attachment of core sugars via the dolichol pyrophosphate pathway.

Structure of Glycosylated and Unglycosylated gag Polyproteins of Rauscher Murine Leukemia Virus: Carbohydrate Attachment Sites

Structure of Glycosylated and Unglycosylated <i>gag</i> Polyproteins of Rauscher Murine Leukemia Virus: Carbohydrate Attachment Sites

Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites

The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15.

Differential uptake of D-galactosyl- and D-glucosyl-neoglycoproteins by isolated rat hepatocytes.

Bovine serum albumin was modified with 2-imino-2-methoxyethyl 1-thioglycopyranosides of galactose and glucose (Gal-AI-BSA, Glc-AI-BSA, respectively). Both types of neoglycoproteins were taken up by isolated rat hepatocytes. Gal-AI-BSA and Glc-AI-BSA inhibited the uptake of each other, and also inhibited the uptake of asialo-orosomucoid as expected from the results obtained with rabbit liver membranes (Stowell, C. P., and Lee, Y. C. (1978) J. Biol. Chem. 253, 6107-6110). Gal-neoglycoproteins of bovine serum albumin containing other linking groups between the galactose moiety and protein were found to be as effective in inhibition of the uptake of asialo-orosomucoid as Gal-AI-BSA, indicating that the method of carbohydrate attachment had little influence on endocytosis of galactose-terminated neoglycoproteins. In contrast, comparable Glc-neoglycoproteins (other than Glc-AI-BSA) were weak inhibitors. It thus appears that the positively charged amidino group present in Glc-AI-BSA contributes to the uptake of this neoglycoprotein by rat hepatocytes.

Complete amino acid sequence of human α 1-microglobulin

Complete amino acid sequence of human α 1-microglobulin has been established. It is composed of 167 amino acid residues and contains three carbohydrate attachment sites. No amino acid sequence heterogeneity was found.

Structure and expression of glycoproteins controlled by the Qa-1a allele.

The alloantigen controlled by the Qa-1a allele is a glycoprotein that exists in two forms. The first, an intracellular molecule of apparent Mr 44 000 daltons, appears to be a kinetic precursor of the second, a cell-surface molecule with an apparent size of 47 000 daltons. The intracellular form of Qa-1 is distinct from that of the TL glycoprotein in two ways: (1) its polypeptide backbone is approximately 5000 daltons shorter, and (2) it possesses three sites of high-mannose carbohydrate attachment, while TL has only one. In the cell-surface form of Qa-1, all three carbohydrate chains are processed to structures that resist endoglycosidase H digestion, presumably complex-type oligosaccharides. Concomitant with these late carbohydrate-processing steps is the formation of stable complexes between Qa-1 and beta 2-microglobulin. The timing of this association provides a further contrast between Qa-1 and TL, which is associated with beta 2-microglobulin shortly after its synthesis. The Qa-1 glycoproteins have been identified genetically by their synthesis in B6-TL+ (Qa-1a/Tlaa) splenocytes but not in splenocytes of congenic B6.K1 and B6.K2 (Qa-1b/Tlab) mice, and by their absence from the products of BALB/c (Qa-1b/Tlac) splenocytes. The cells synthesizing Qa-1 are at least as prevalent in Ig+ spleen cell populations as in T-cell-enriched splenic Ig- populations. Thus, active Qa-1 synthesis appears to take place at a high rate in normal splenic B cells without mitogenic stimulation.

Covalent structure of turnip peroxidase 7. Tryptic peptides.

Turnip isoperoxidase TP 7 had its hemin group removed and was cleaved by trypsin. The digest was fractionated by gel filtration and high-voltage paper electrophoresis and yielded 24 peptides, which counted for all 296 amino acid residues of the enzyme. Sequence analyses on the tryptic peptides and, when necessary, on their thermolytic derivatives were carried out by manual Edman degradation followed by dansylation or by quantitative amino acid analysis of thiazolinones converted by HI. The four disulfide bridges and the only site of carbohydrate attachment of turnip peroxidase 7 are located. The present analysis of tryptic peptides has been complemented by cyanogen bromide fragments cleaved by chymotrypsin as described in the accompanying paper.

Primary structures of pancreatic ribonucleases from Bovidae: Impala, Thomson's gazelle, nilgai and water buffalo

The amino acid sequences of the pancreatic ribonucleases from impala, Thomson's gazelle, nilgai and two types of water buffalo were studied from several enzymic digests. Peptides were positioned by homology with other ribonucleases of known sequence. Only peptides that differ in amino acid composition from the corresponding peptides of ox and goat ribonuclease were sequenced. The primary structures of pancreatic ribonucleases from 11 species of the Bovidae family are known to date. The evolutionary rate of bovid ribonucleases is 2–3 times lower than the average rate observed in all mammals. A possible explanation is that the presence of a stable symbiotic system in the rumen of grass-eating ruminants has caused a slowing down of the evolutionary rate of pancreatic ribonuclease in this taxon. The subfamily of the Bovinae (five species) exhibits a slightly higher evolutionary rate; most replacements in the Bovinae occur near residues 34–36, the most commonly observed carbohydrate attachment site in ribonucleases.

The partial primary structure of bovine rhodopsin and its topography in the retinal rod cell disc membrane

The amino-terminal 39 amino acids of bovine rhodopsin have the sequence ▪ where both carbohydrate attachment sites (CHO) contain GlcNAc 3Man 3. This region of rhodopsin's sequence is exposed at the internal membrane surface of the rod cell disc membrane. Rhodopsin's carboxyl-terminal 40 amino acids have the sequence ▪ where amino acid 1′ is the carboxyl-terminal amino acid of rhodopsin. Serines and threonines in the sequence 6′ → 15′ are phosphorylated by rhodopsin kinase in a light-dependent reaction. Trypsin can digest native rhodopsin, in the disc membrane at ▪ and thermolysin can hydrolyze bonds ▪, and ▪. Limited proteolysis by thermolysin at a site internal in the molecule has been exploited in order to prepare rhodopsin as two large fragments, F1 and F2. Cysteine 33′, is highly reactive in the dark and is modified by N-ethylmaleimide and several alkylating agents. The carboxyl-terminal region 1′–39′ reacts with the membrane-impermeable nitrene from N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate and is therefore exposed at the external (cytoplasmic) surface of the disc membrane. 1-azldopyrene, a hydrophobic nitrene precursor, is being used to map those regions of the rhodopsin sequence which are located in a hydrophobic environment.

Structural studies of human IgD: Isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation

A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD delta chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the delta chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc(delta) by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the delta-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.

Resolution of the charge forms and amino acid sequence and location of a tryptic glycopeptide in rat alpha-lactalbumin.

Three charge forms of rat alpha-lactalbumin were separated by ion exchange chromatography on DEAE-cellulose. The amino acid composition of each form was similar but they differed in carbohydrate composition. Each form contained a tryptic glycopeptide having a common polypeptide and heteropolysaccharide unit. The tryptic glycopeptide was sequenced and positioned in rat alpha-lactalbumin, which was partially sequenced from residues 1 to 50. The carbohydrate attachment site was at Asn45. Secondary structure calculations predicted that Asn45 is in a beta bend conformation whereas Asn45 in bovine alpha-lactalbumin, a poorly glycosylated protein, is not in a bend conformation.

Transfer of carbohydrates on to nascent glycoprotein of vesicular stomatitis virus

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.

Process of Iodination of Thyroglobulin and Its Maturation&lt;subtitle&gt;II. Properties and Distribution of Thyroglobulin Labeled &lt;italic&gt;In Vitro&lt;/italic&gt; or &lt;italic&gt;In Vivo&lt;/italic&gt; with Radioiodine, &lt;sup&gt;3&lt;/sup&gt;H-Tyrosine, or &lt;sup&gt;3&lt;/sup&gt;H-Galactose in Rat Thyroid Glands&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/subtitle&gt;

With the aim of obtaining information on the process of iodination of thyroglobulin, the properties and subcellular distribution of thyroglobulin labeled with radioiodine, 3H-tyrosine, or 3H-galactose were studied. The following results were obtained for 17-19S thyroglobulin isolated from rat thyroid lobes labeled in vitro. (a) The effect of sodium dodecyl sulfate (SDS) concentration (0.1-2.0 mM) on the dissociability of the proteins into 12S subunits showed that 3H-labeled, 131I-labeled, and preformed thyroglobulin behaved very differently; their dissociability decreased in that order. In addition, 0.3 mM SDS is most suitable for discriminating among these species. (b) The amount of 0.3 mM SDS-resistant 131I-thyroglobulin increased with the time of incubation of the lobes or with the amount of iodine atoms incorporated by chemical iodination. (c) Digestion of 3H-tyrosine-labeled thyroglobulin showed that 3H-monoiodotyrosine and 3H-diiodotyrosine were present after incubation of the lobes for 180 min. (d) The dissociability of 3H-galactose-labeled 17-19S thyroglobulin was higher than that of 131I-labeled protein, but its elution pattern on DEAE-cellulose chromatography resembled that of the latter. (e) 131I-Thyroglobulin was scarcely found in the incubation medium, although a considerable amount of 19S thyroglobulin was released into the medium during the incubation. As for the lobes, a significant amount of 131I-radioactivity as well as 3H-radioactivity was found in cytoplasmic particulates, especially in fractions containing apical vesicles and rough microsomes. On the other hand, the following results were obtained for 17-19S thyroglobulin isolated from rats injected with 125I. (a) Dissociability of the protein by 0.3 mM SDS and analysis of 125I-iodoamino acids of pronase digest showed that the iodination process was essentially similar to the case of in vitro incorporation, but was faster. (b) The effect of cyclohiximide treatment showed that the relative reduction of 0.3 mM SDS dissociable species was probably due to a shortage of newly synthesized proteins. All the results obtained in the present experiments are compatible with the view that iodine atoms are incorporated selectively into newly synthesized, less iodinated thyroglobulin, and that the iodination occurs intracellularly, at least to a certain degree, after carbohydrate attachment, probably in the apical vesicles. The possibility that iodination also occurs to some extent in the endoplasmic reticulum and in the colloid lumen of thyroglobulin-stimulated thyroids is discussed.

Amino acid sequence of a mouse immunoglobulin mu chain.

The complete amino acid sequence of the mouse mu chain from the BALB/c myeloma tumor MOPC 104E is reported. The C mu region contains four consecutive homology regions of approximately 110 residues and a COOH-terminal region of 19 residues. A comparison of this mu chain from mouse with a complete mu sequence from human (Ou) and a partial mu chain sequence from dog (Moo) reveals a striking gradient of increasing homology from the NH2-terminal to the COOH-terminal portion of these mu chains, with the former being the least and the latter the most highly conserved. Four of the five sites of carbohydrate attachment appear to be at identical residue positions when the constant regions of the mouse and human mu chains are compared. The mu chain of MOPC 104E has a carbohydrate moiety attached in the second hypervariable region. This is particularly interesting in view of the fact that MOPC 104E binds alpha-(1 leads to 3)-dextran, a simple carbohydrate. The structural and functional constraints imposed by these comparative sequence analyses are discussed.